Journal: Infection and Immunity

Article Title: NF-κB RelA Is Required for Hepatoprotection during Pneumonia and Sepsis

doi: 10.1128/IAI.00132-19

Figure Lengend Snippet: Hepatoprotection during pneumonia is RelA dependent. E. coli (106 CFU) was instilled i.t. into 15-week-old mice lacking either NF-κB RelA (hepRelAΔ/Δ mice) (A) or STAT3 (hepSTAT3Δ/Δ mice) (B) in hepatocytes, and the results were compared to those for the littermate controls (WT). Survival over a 24-h period is illustrated. Liver injury was assessed by determination of serum ALT and AST concentrations 24 h after i.t. E. coli challenge (n = 4 to 6 per group). (C) Blood bacterial burdens were quantified in mice 24 h after i.t. E. coli challenge. (D) Serum ALT levels were compared in mice with and without detectable bacteremia 24 h after i.t. E. coli challenge. *, P < 0.05 versus WT (n = 3 to 4 per group).

Article Snippet: Briefly, a 24-gauge catheter was inserted into the exposed trachea, and a 50-μl bolus of saline containing 10 6 CFU of Escherichia coli (serotype O6:K2:H1, ATCC 19138), 10 3 CFU of Klebsiella pneumoniae (ATCC 43816), or 10 6 CFU of Streptococcus pneumoniae (serotype 3, ATCC 6303) was instilled into the left bronchus.

Techniques:

Journal: Infection and Immunity

Article Title: NF-κB RelA Is Required for Hepatoprotection during Pneumonia and Sepsis

doi: 10.1128/IAI.00132-19

Figure Lengend Snippet: RelA-dependent hepatoprotection extends beyond pneumonia. Serum ALT concentrations were measured in hepRelAΔ/Δ or WT mice challenged with i.t. K. pneumoniae (103 CFU) (A), i.t. S. pneumoniae (106 CFU) (B), i.v. E. coli (106 CFU) (C), i.v. S. pneumoniae (106 CFU) (D), i.v. heat-killed S. pneumoniae (107 CFU) (E), or i.p. lipopolysaccharide (LPS; 0.1 mg/kg) (F). *, P < 0.05 versus WT (n = 3 to 12 per group). (G) Livers were collected 24 h after i.v. E. coli challenge and processed for histological analysis by hematoxylin and eosin staining. Representative images are shown for each genotype. Bar, 75 μm. MFI, mean fluorescence intensity.

Article Snippet: Briefly, a 24-gauge catheter was inserted into the exposed trachea, and a 50-μl bolus of saline containing 10 6 CFU of Escherichia coli (serotype O6:K2:H1, ATCC 19138), 10 3 CFU of Klebsiella pneumoniae (ATCC 43816), or 10 6 CFU of Streptococcus pneumoniae (serotype 3, ATCC 6303) was instilled into the left bronchus.

Techniques: Staining, Fluorescence

Journal: Infection and Immunity

Article Title: NF-κB RelA Is Required for Hepatoprotection during Pneumonia and Sepsis

doi: 10.1128/IAI.00132-19

Figure Lengend Snippet: Liver injury in hepRelAΔ/Δ mice is associated with apoptosis. (A to C) hepRelAΔ/Δ or WT mice were challenged with i.t. E. coli (106 CFU) for 16 h (A), i.t. K. pneumoniae (103 CFU) for 48 h (B), or i.v. S. pneumoniae (106 CFU) for 24 h (C). Livers were collected, and the presence of active caspase-3 and -8 in liver tissue homogenates was determined by immunoblotting. The results of densitometry analysis are shown as caspase/pan-actin ratios. (D) The presence of phospho-MLKL was determined by immunoblotting of whole-liver-tissue homogenates collected from hepRelAΔ/Δ or WT mice 0 and 24 h after i.v. S. pneumoniae challenge. (E and F) Serum ALT concentrations (E) and the presence of phospho-MLKL (F) were measured in hepRelAΔ/Δ mice pretreated with Nec-1s or vehicle and challenged with i.v. heat-killed S. pneumoniae for 24 h. *, P < 0.05 versus WT (n = 3 per group). Cl., cleaved.

Article Snippet: Briefly, a 24-gauge catheter was inserted into the exposed trachea, and a 50-μl bolus of saline containing 10 6 CFU of Escherichia coli (serotype O6:K2:H1, ATCC 19138), 10 3 CFU of Klebsiella pneumoniae (ATCC 43816), or 10 6 CFU of Streptococcus pneumoniae (serotype 3, ATCC 6303) was instilled into the left bronchus.

Techniques: Western Blot

Journal: Infection and Immunity

Article Title: NF-κB RelA Is Required for Hepatoprotection during Pneumonia and Sepsis

doi: 10.1128/IAI.00132-19

Figure Lengend Snippet: NKT cell activity is sufficient but not necessary for hepatotoxicity in hepRelAΔ/Δ mice. (A) C57BL/6 mice were challenged i.v. with 106 CFU of E. coli for 24 h. Single-cell suspensions of mononuclear cells were generated from whole liver tissues. NKT cells (CD45+/CD3+/α-galactoceramide [αGalCer]-loaded CD1d tetramer positive) were identified by flow cytometry, and CD69+ surface expression and mean fluorescence intensity were measured as an activation marker. n = 3 per group; *, P < 0.05 versus saline. (B and C) Serum ALT was measured in hepRelAΔ/Δ or WT mice challenged i.v. for 24 h with 2 μg α-GalCer or vehicle (n = 3 to 6 per group; *, P < 0.05 versus vehicle hepRelAΔ/Δ) (B) and in αGalCer-treated mice coinstilled with control IgG or anti-TNF-α neutralizing antibody (n = 3 to 6 per group; *, P < 0.05 versus control IgG hepRelAΔ/Δ) (C). (D) Blood bacterial burdens were measured 7 h after i.v. challenge with 106 CFU of E. coli in C57BL/6 mice treated with control IgG or an anti-CD1d antibody blocking NKT cell activity. *, P < 0.05 versus IgG. (E) Serum ALT was measured in hepRelAΔ/Δ or WT mice challenged i.v. with 107 CFU of heat-killed S. pneumoniae for 24 h. n = 3 to 6 per group; *, P < 0.05 versus WT.

Article Snippet: Briefly, a 24-gauge catheter was inserted into the exposed trachea, and a 50-μl bolus of saline containing 10 6 CFU of Escherichia coli (serotype O6:K2:H1, ATCC 19138), 10 3 CFU of Klebsiella pneumoniae (ATCC 43816), or 10 6 CFU of Streptococcus pneumoniae (serotype 3, ATCC 6303) was instilled into the left bronchus.

Techniques: Activity Assay, Generated, Flow Cytometry, Expressing, Fluorescence, Activation Assay, Marker, Saline, Control, Blocking Assay

Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: PRL3-zumab inhibits PRL3 + liver tumors in vivo but not cancer cells in vitro. a Representative western blot (WB) of PRL3 protein expression in human (lanes 1–6) and murine (lanes 7 and 8) liver cancer cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. Asterisks indicate cell lines that rapidly generate orthotopic liver tumors within 5 weeks. b Outline of orthotopic “seed and soil” liver tumor model for treatments. c – e Mean volumes at the end of the experiment in treated (filled squares) and untreated (filled triangles) groups of mice bearing PRL3 + MHCC-LM3 tumors ( n = 10 mice per group; c ), PRL3 − Hep53.4 tumors ( n = 3 mice per group; d ), and Hep53.4-PRL3 tumors ( n = 6 mice per group; e ). The mean value was calculated by the Student’s t test (mean ± s.e.m.). P values between treatment pairs as indicated. Lower panels, representative liver tumors at the end of experiment. Scale bar, 10 mm. f – h The viabilities of MHCC-LM3 cells ( f ), Hep53.4 cells ( g ), and Hep53.4-PRL3 cells ( h ) cultured for 48 h with PBS control (filled squares), 5 µg mL −1 PRL3-zumab (filled upright triangles), 50 µg mL −1 PRL3-zumab (filled inverted triangles), 2 µg mL −1 cisplatin (filled diamonds), or 10 µg mL −1 cisplatin (filled circles) were evaluated by an MTS (3-(4,5-dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)-2 H -tetrazolium) assay. The mean value was calculated by the Student’s t test (mean ± s.e.m., n = 3 biologically independent samples each). * P < 0.05, ** P < 0.01, NS, not significant, as compared between treatment and control group for each cell line. Source data are provided as a Source Data file

Article Snippet: The Hep53.4-PRL3 cell line, which stably expresses EGFP-tagged PRL3 fusion protein, was generated by using jetPRIME reagent (Polyplus-transfection) to transfect Hep53.4 cells with the pEGFP-C1-PRL-3 plasmid .

Techniques: In Vivo, In Vitro, Western Blot, Expressing, Cell Culture

Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: Intracellular PRL3 is externalized for PRL3-zumab binding. a Methodology for cell surface analysis of MHCC-LM3 cultured cells (CC) and tumor cells. b – d “Surface” detection of nonspecific control antigens ( b ), EGFR ( c ), or PRL3 ( d ) were detected by fluorescence-activated cell sorting (FACS) analysis using polyclonal human hIgG, anti-EGFR antibody (cetuximab), or anti-PRL3 antibody (PRL3-zumab), respectively. Representative FACS profiles from four biological replicates are shown. e Mean percentage ± s.d. of surface positive (surface+) live cells for each antigen were calculated by dividing the surface antigen-positive live cells (upper left quadrant) by total live cells (sum of both upper and lower left quadrants) in b – d . f Background-corrected values from e were normalized to CC surface expression levels for EGFR (filled circles) and PRL3 (filled squares). The mean fold change was calculated by the Student’s t test (mean ± s.d., n = 4 biologically independent samples). P values as indicated for each antigen. g Background-corrected values of MHCC-LM3 cells cultured under “Normal” vs. “Serum-starved” conditions for 72 h were normalized to “Normal” surface+ cell percentages for each antigen. The mean fold-change was calculated by the Student’s t test (mean ± s.d.) for EGFR (filled circles; n = 3 independent samples) and PRL3 (filled squares; n = 4 independent samples). P values as indicated for each antigen. Source data are provided as a Source Data file

Article Snippet: The Hep53.4-PRL3 cell line, which stably expresses EGFP-tagged PRL3 fusion protein, was generated by using jetPRIME reagent (Polyplus-transfection) to transfect Hep53.4 cells with the pEGFP-C1-PRL-3 plasmid .

Techniques: Binding Assay, Cell Culture, Fluorescence, FACS, Expressing

Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: PRL3 is expressed on the surface of exosomes from PRL3 + cancer cells. a Representative PRL3 western blot (WB) in purified exosomes from MHCC-LM3 and Hep53.4 liver cancer cells cultured for 24 h in serum-free media. Paxillin served as a negative marker for exosomes, whereas TSG101 is a positive exosomal marker. b Immunofluorescence analysis of MHCC-LM3 cells transiently transfected with green fluorescence protein (GFP) or GFP-PRL3 to reveal plasma membrane enrichment of PRL3. Scale bar, 20 µm. c Representative PRL3 WB in purified exosomes from cells in b after culturing for 24 h in serum-free media. Calnexin served as a negative marker for exosomes. d Purified exosomes from Hep53.4 and Hep53.4-PRL3 were analyzed as in c . e Purified exosomes from cells in c , d were assayed for sensitivity of exosomal PRL3 to proteinase K (Prot. K) digestion in the absence or presence of 1% Triton X-100 detergent. TG101 and Alix are intravesicular exosomal proteins. f Proposed model of the localization of PRL3, TSG101, and Alix in exosomes, based on Prot. K susceptibility observed in e . Source data are provided as a Source Data file

Article Snippet: The Hep53.4-PRL3 cell line, which stably expresses EGFP-tagged PRL3 fusion protein, was generated by using jetPRIME reagent (Polyplus-transfection) to transfect Hep53.4 cells with the pEGFP-C1-PRL-3 plasmid .

Techniques: Western Blot, Purification, Cell Culture, Marker, Immunofluorescence, Transfection, Fluorescence, Membrane

Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: PRL3-zumab eliminates tumors in an Fc- and FcR-dependent manner. a Cartoon depicting domain architecture of PRL3-zumab (intact Fc) vs. PRL3-minibody (truncated Fc lacking CH1 and CH2 domains) and their ability to engage Fc receptors (FcR) on host immune cells. The anti-CD16/32 FcR blocker antibody prevents IgG from binding murine FcγII/III receptors. b Both Fc region of PRL3-zumab and FcR binding are required for anti-tumor effects of PRL3-zumab. Upper panel, representative images of livers from each treatment group at day 35 (5-week endpoint). Tumor areas are framed with black lines. Scale bar, 10 mm. Lower panel, tumor volumes in each group. The mean tumor volumes were calculated using one-way analysis of variance (ANOVA) (mean ± s.e.m.) for independent groups of untreated ( n = 12), human IgG-treated ( n = 3), PRL3-zumab-treated ( n = 6), PRL3-minibody-treated ( n = 5), PRL3-zumab + FcR blocked-treated ( n = 7), and FcR blocker-treated ( n = 4) mice. Source data are provided as a Source Data file

Article Snippet: The Hep53.4-PRL3 cell line, which stably expresses EGFP-tagged PRL3 fusion protein, was generated by using jetPRIME reagent (Polyplus-transfection) to transfect Hep53.4 cells with the pEGFP-C1-PRL-3 plasmid .

Techniques: Binding Assay

Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: PRL3-zumab recruits immune cells to tumor sites in an FcR-dependent manner. a – c Orthotopic MHCC-LM3 liver tumor tissue cryo-sections from various groups of mice: I) untreated (filled black squares), II) PRL3-zumab treatment (filled red triangles), III) FcR blocker treatment (filled black triangles), and IV) PRL3-zumab plus Fc blocker combination treatment (filled black diamonds) were analyzed by immunofluorescence with antibodies against B220/CD45R (B cells; a ), CD335 (NK cells; b ), or F4/80 (macrophages; c ) and scored for relative tumor infiltration. The mean relative infiltration was calculated using one-way analysis of variance (ANOVA) (mean ± s.e.m., n = 4 independent samples). Lower panels, representative immunofluorescence results for each antibody set. Scale bar, 200 µm. d Proposed mechanism of action of PRL3-zumab. Externalized PRL3 antigens are recognized by PRL3-zumab, which then recruits NK cells, B cells, and Ly-6C + F4/80 + macrophages for tumor killing and leakage of additional PRL3 antigens (“kill-and-leak” cycle) triggering an anti-tumor ADCC/ADCP cascade. Source data are provided as a Source Data file

Article Snippet: The Hep53.4-PRL3 cell line, which stably expresses EGFP-tagged PRL3 fusion protein, was generated by using jetPRIME reagent (Polyplus-transfection) to transfect Hep53.4 cells with the pEGFP-C1-PRL-3 plasmid .

Techniques: Immunofluorescence

Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: PRL3 is frequently overexpressed in multiple human cancers. a – k Representative full western blot (WB) of PRL3 protein levels in tumor (“T”) tissues and, where available, patient-matched normal (“n”) tissues from a liver, b lung, c colon, d breast, e stomach, f thyroid, g pancreas, h kidney, i acute myeloid leukemia (bone marrow aspirates), j bladder, and k prostate tissues. Relative molecular masses (in kDa) are indicated on the right of each immunoblot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. Source data are provided as a Source Data file

Article Snippet: The Hep53.4-PRL3 cell line, which stably expresses EGFP-tagged PRL3 fusion protein, was generated by using jetPRIME reagent (Polyplus-transfection) to transfect Hep53.4 cells with the pEGFP-C1-PRL-3 plasmid .

Techniques: Western Blot

Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: Summary of PRL3 expression across different tumor types

Article Snippet: The Hep53.4-PRL3 cell line, which stably expresses EGFP-tagged PRL3 fusion protein, was generated by using jetPRIME reagent (Polyplus-transfection) to transfect Hep53.4 cells with the pEGFP-C1-PRL-3 plasmid .

Techniques: Expressing

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Graphic for the ultrastructural parameters analyzed in electron micrographs for GluA2-His and neurexin1-His expressing neurons. ( B) Representative electron micrographs of cryofixed primary hippocampal neurons expressing GluA2-His or neurexin1-His. ( C and E) Violin, and cumulative probability plots of gold particle distribution at the normalized AZ. ( D and G) Violin, and cumulative probability plots of the nearest distance between gold participles and the next docked SV compared to randomized distances (dashed line). Insert depicts the residual percentage of the distance of gold particles to the next docked SV compared to their randomized distribution. ( E and H) Violin, and cumulative probability plots of the measured data and null model data of mean nearest neighbor distance (NND) to docked SVs. Data for violin plots are medians (solid lines) and quartiles (dashed lines). Statistical significance for violin plots in C, D, E, F, G, and H was assessed by the Mann-Whitney test and for the cumulative distribution plots C, D, E, F, G, and H by Kolmogorov-Smirnov test. *p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Graphic for the ultrastructural parameters analyzed in electron micrographs for GluA2-His, neurexin-His, and neuroligin-His expressing neurons. ( B) Representative electron micrographs of cryofixed primary hippocampal neurons expressing GluA2-HIS, neurexin1-His, or neuroligin1-His. ( C) Summary graph of docked SV number per AZ. ( D) Summary graph of the PSD length. ( E) Cumulative probability of docked SV distribution at the AZ. ( F) Summary graph of gold particles per AZ. ( G) Cumulative probability plot of gold particles per AZ. h) Relative frequency of gold particles in the synaptic cleft (0 = active zone membrane; 1 = post-synaptic membrane). ( I) Violin, and cumulative probability plots of gold the particle distribution at the normalized AZ for neuroligin-His expressing neurons compared to randomized data. ( J) Violin, and cumulative probability plots of the nearest distance between gold participles and the next docked SV compared to randomized distances. ( K) Violin, and cumulative probability plots of the measured data and null model data of mean nearest neighbor distance (NND) of gold particles to docked SVs. Data for bar graphs are individual values and means ± SEM. Data for violin plots are medians (solid lines) and quartiles (dashed lines). Statistical significance for C, D, and F was assessed by Kruskal-Wallis test, for I, J, and K by Mann-Whitney test, and for the cumulative distribution plots in I, J, and K by the Kolmogorov-Smirnov test. *p < 0.05.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A-H) , Experiments were conducted in RIM/RBP deficient synapses (qKO) and their respective control (Δcre). ( A and C) Representative electron micrographs of cryo-fixed control and qKO primary hippocampal neurons expressing either GluA2-His or α2δ1-His. Scale bar, 200 nm. ( B and D) Number of gold particles per AZ. (E) Synaptic cleft width. ( F) Representative dSTORM images of GluA1 and GluA2 (green) and Homer (red) expressed in control and qKO neurons. Scale bars 1 µm. ( G) Number of GluA1 clusters per synapse area. ( H) Number of GluA2 clusters per synapse area. ( I) Sample traces of mEPSC events in control (grey) and qKO (red) derived from autaptic neurons. ( J) Miniature excitatory postsynaptic current (mEPSC) amplitude. ( K) mEPSC rise time. Data are individual values and means ± SEM. Statistical significance for B, D, E, J, K, G, and H was assessed by unpaired t-test and cumulative distribution plots in B and D by Kolmogorov-Smirnov test. *p < 0.05, ***p < 0.001.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Control, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Model of transsynaptic alignment of α2δ1-AP and GluA2-mSA by BirA co-expression in qKO synapses. ( B) Example traces of excitatory postsynaptic currents (EPSC). ( C) Excitatory postsynaptic current (EPSC) amplitudes. ( D) Example traces of synaptic responses to a 5 s application of hypertonic sucrose. ( E) Readily releasable pool (RRP) charge. ( F) Representative electron micrographs of cryofixed primary hippocampal neurons from qKO mice. ( G) Absolute number of docked SV and docked SV per 100 nm AZ. ( H) Example images of SynGCaMP6f fluorescence in qKO neurons, obtained during trains of AP stimulation. Scale bars 5 µm ( I) Fluorescence changes (ΔF/F) upon single AP and APs trains. Data are individual values and means ± SEM. Statistical significance for C, E, and G was assessed by Kruskal-Wallis test and I by Two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Representative electron micrographs of cryofixed primary hippocampal neurons from qKO mice expressing α2δ1-AP and GluA2-mSA or α2δ1-AP, GluA2-mSA, and BirA. Scale bars 100 nm ( B) Cumulative probability plot and bar graph of the synaptic cleft width. ( C) Cumulative probability plot and bar graph of the PSD length. ( D) Example traces of miniature postsynaptic currents (mEPSCs).( E) Cumulative probability plot, and bar graph of mEPSC frequency. ( F) Cumulative probability plot, and bar graph of mEPSC amplitude. ( G) Cumulative probability plot, and bar graph of mEPSC rise time. ( H) Example images of Munc13-1 and synaptophysin immunostainings in neuronal cultures obtained from qKO mice. Scale bars 5 µm. ( I) Munc13-1 immunofluorescence intensities normalized to synaptophysin intensities. Data are means ± SEM. Statistical significance for B, C, E, F, G, and I was assessed by Kruskal-Wallis test. *p < 0.05, **p < 0.01.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, Immunofluorescence

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, phase contrast (left panel) and fluorescence micrographs (right panel) of NIH/3T3 cell transfected with gpClC-3-GFP. B, representative IgpClC-3 recorded from GFP-positive NIH/3T3 cells (as shown in A) under isotonic, hypotonic and hypertonic conditions over the range -100 to +120 mV. C, effects of anti-ClC-3 Ab on IgpClC-3 at ±80 mV when pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). D, mean current densities recorded from untransfected NIH/3T3 cells (blue, n = 38), gpClC-3-GFP-transfected cells dialysed with standard intracellular pipette solutions (green, n = 9), preabsorbed anti-ClC-3 Ab (black, n = 4), and anti-ClC-3 Ab alone (red, n = 9).

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Fluorescence, Transfection, Transferring

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, time course of VSOAC currents from two PASMCs dialysed with either anti-ClC-3 Ab (5 μg ml−1) preabsorbed with antigen (50 μg ml−1; ○), or anti-ClC-3 Ab alone (5 μg ml−1; •). Inset: Western blot analysis of native ClC-3 expression in isolated canine PASMCs. B, mean current densities in cells dialysed with either standard intracellular solutions (n = 11), preabsorbed anti-ClC-3 Ab (n = 4) or anti-ClC-3 Ab alone (n = 5).

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Western Blot, Expressing, Isolation

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions. Aa, pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). Ab, mean current densities for preabsorbed anti-ClC-3 Ab (□, n = 3) or anti-ClC-3 Ab alone (▪, n = 5) at times indicated. B, same as A, except cells were preswelled by pre-exposure to hypotonic bath solutions prior to membrane rupture and dialysis; preabsorbed anti-ClC-3 Ab (□, n = 4), and anti-ClC-3 Ab alone (▪, n = 5).

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Transferring

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, Western blot analysis of native ClC-3 expression in oocytes. B, 4-phorbol 12,13-dibutyrate (PDBu) inhibition of native VSOAC currents in oocytes (-100 to +120 mV). C, time course of VSOAC activation in response to hypotonic bath solutions from a non-injected control oocyte (○) and an oocyte injected with anti-ClC-3 Ab (15 μg ml-1; •) at time shown. Cells were held at -30 mV for 30 ms, hyperpolarized to -100 mV for 210 ms and then depolarized to +100 mV for 210 ms, repetitively at 2 Hz. D, mean current densities from control oocytes (n = 10), oocytes injected with anti-ClC-3 Ab alone (15 μg ml-1) (n = 4) or injected with preabsorbed anti-ClC-3 Ab (150 μg ml-1) (n = 4). Peak current densities were measured after 5 min exposure to isotonic solutions, and after 85 min exposure to hypotonic solutions.

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Western Blot, Expressing, Inhibition, Activation Assay, Injection